Our long range goal is to define the role which calcium plays in early events leading to renal ischemic cell injury. To do so, it is vital to identify the sites of release, uptake and binding of calcium within the cells. Electron probe x-ray microanalysis (EPXMA) offers and unexcelled means of measuring elemental contents within individual cells and subcellular organelles, so that changes in the amounts of localized calcium can be quantitated at chosen intervals during the time course leading to irreversible cell injury. Preliminary experiments using EPXMA in proximal tubules have demonstrated a large degree of heterogeneity in the cellular and intracellular distribution of calcium. Such heterogeneity brings into question numerous previous studies which have assumed that there is a more homogeneous distribution. The proximal tubule suspension facilitates experimental maneuvers which permit examination of the earliest events leading to irreversible cell damage and eliminates secondary effects that, in an intact animal model, arise from compromising the function of the renovascular system in toto. We shall perform, in parallel, experiments which will measure biochemical, physiological, ultrastructural and microchemical variables on the same suspension. Experimental conditions will also be examined that preserve renal function during anoxia, such as the presence of adenine nucleotides, mannitol, and low pH in the bathing medium. Experiments will be performed to measure cytosolic free calcium using methyl ester dyes (fura-2) under identical conditions. EPXMA will be used to validate these measurement by determining the compartmentation of the dyes. This powerful technique (EPXMA) judiciously applied to a well-characterized renal preparation, in combination with the established array of physiological, biochemical, and ultrastructural measurements, is expected to provide unique direct information on the role of calcium in hypoxic cell injury in the kidney.